ABSTRACT
BACKGROUND: The occurrence and prevalence of integrons in clinical microorganisms and their role played in antimicrobial resistance have been well studied recently. As screening and detection of integrons are concerned, current diagnostic methodologies are restricted by significant drawbacks and novel methods are required for integrons detection. RESULTS: In this study, three loop-mediated isothermal amplification (LAMP) assays targeting on class 1, 2 and 3 integrons were implemented and evaluated. Optimization of these detection assays were performed, including studing on the reaction temperature, volume, time, sensitivity and specificity (both primers and targets). Application of the established LAMP assays were further verified on a total of 1082 isolates (previously identified to be 397 integron-positive and 685 integron-negative strains). According to the results, the indispensability of each primer had been confirmed and the optimal reaction temperature, volume and time were found to be 65°C, 45 min and 25 µL, respectively. As application was concerned, 361, 28 and 8 isolates carrying intI1, intI2 and intI3 yielded positive amplicons, respectively. Other 685 integron-negative bacteria were negative for the integron-screening LAMP assays, totaling the detection rate and specificity to be 100%. CONCLUSIONS: The intI1-, intI2- and intI3-LAMP assays established in this study were demonstrated to be the valid and rapid detection methodologies for the screening of bacterial integrons.
Subject(s)
DNA, Bacterial/isolation & purification , Nucleic Acid Amplification Techniques/methods , Integrons , Organic Chemicals , Salmonella/genetics , Serratia marcescens/genetics , Staphylococcus/genetics , Vibrio cholerae/genetics , Colony Count, Microbial , Microbial Sensitivity Tests , Polymerase Chain Reaction/methods , Sensitivity and Specificity , DNA, Complementary , DNA Primers , Integrases/genetics , Drug Resistance, Bacterial/genetics , Electrophoresis, Agar Gel , Escherichia coli/genetics , Fluorescent Dyes , Hot TemperatureABSTRACT
<p><b>OBJECTIVE</b>To investigate the effects of Trichophyton rubrum exposure on the expressions of toll-like receptor-2 (TLR-2), TLR-4 and dendritic cell associated C-type lectin-1 (Dectin-1) and cytokine secretions in human keratinocytes cell line HaCaT.</p><p><b>METHODS</b>The mRNA of TLR-2,4, and dectin-1 in the HaCaT co-cultured with the conidia of Trichophyton rubrum conidia for 24 h was measured with real-time PCR. The mean fluorescence intensity and the percentage of cells positive for TLR-2, 4, and dectin-1 was detected during the co-culture using flow cytometry. The cytokine secretion profiles in the cell culture supernatant was analyzed using a cytokine antibody array.</p><p><b>RESULTS</b>The TLR-2,4, and dectin-1 mRNA expressions, mean fluorescence intensity and percentage of positive cells for TLR-2,4, and dectin-1 all increased in HaCaT cells in response to Trichophyton rubrum conidia exposure. The results of cytokine antibody array demonstrated obviously increased secretions of IL-8, I-309, IFN-γ, IL-6, and IL-13 in the culture supernatant of HaCaT cells in response to Trichophyton rubrum exposure.</p><p><b>CONCLUSION</b>The immune responses and immunological recognition of human keratinocytes to Trichophyton rubrum conidia are partially mediated by up-regulating the expressions of TLR-2, TLR-4 and dectin-1 and secretions of multiple cytokines.</p>
Subject(s)
Humans , Cell Line , Chemokine CCL1 , Bodily Secretions , Coculture Techniques , Interferon-gamma , Bodily Secretions , Interleukin-13 , Bodily Secretions , Interleukin-6 , Bodily Secretions , Interleukin-8 , Bodily Secretions , Keratinocytes , Metabolism , Lectins, C-Type , Metabolism , Toll-Like Receptor 2 , Metabolism , Toll-Like Receptor 4 , Metabolism , Trichophyton , Tumor Necrosis Factor-alpha , Bodily SecretionsABSTRACT
<p><b>BACKGROUND</b>Cathepsin B plays an important role in cell cycle, extracellular matrix changes and cutaneous tumorigenesis: whether it plays a role in photoaged skin remains unknown. This study aimed to investigate the role of cathepsin B in skin photoaging in vivo and in vitro.</p><p><b>METHODS</b>The expressions of cathepsin B were compared with immunohistochemical methods in solar exposed skin and solar protected skin of six healthy Chinese volunteers. The mRNA and protein expression of cathepsin B in ultraviolet light A (UVA) induced premature senescence fibroblasts in vitro were detected by real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blotting technique.</p><p><b>RESULTS</b>Decreased expression of cathepsin B was observed in photoaged skin compared with that of the solar protected skin. In the UVA induced, premature senescence fibroblasts, a lower expression of cathepsin B was detected by Western blotting and a decreased synthesis of cathepsin B mRNA in the same cells was revealed by real-time RT-PCR.</p><p><b>CONCLUSIONS</b>The results demonstrated a significant negative correlation between skin photoaging and cathepsin B in vitro and in vivo. We propose that cathepsin B, besides matrix metalloproteinases and antioxidant enzymes, is involved in the process of skin photoaging in that it contributes to extracellular matrix remodelling and is a dominant protease in cellular apoptosis and senescence.</p>